Cell-cell communication by acyl-homoserine lactone (acyl-HSL) quorum sensing (QS) is common to a variety of Gram-negative Proteobacteria and regulates diverse biological functions. QS involves acyl-HSL production and subsequent detection by a community of bacteria in order to monitor their cell density. Acyl-HSLs are detected by transcriptional regulators, which affect global changes in gene expression. QS is important for virulence in many organisms, including the category B bioagent Burkholderia mallei, the causative agent of glanders. Disruption of one of the B. mallei acyl-HSL receptors, BmaR5, severely impairs virulence in mice and hamsters. These observations lead to the hypothesis that this QS receptor controls transcriptional regulation of important virulence genes, and that inhibition of BmaRS will block this crucial regulation. Acyl-HSL receptor genes are commonly linked to acyl-HSL synthase genes, but BmaR5 represents a subgroup of receptors called orphans because there is no linked acyl-HSL synthase gene. The biological significance of orphan receptors is not well understood. The aims of this application are to characterize BmaR5 by identifying the acyl-HSL signal to which it responds, determining and characterizing promoter targets of this protein, and finding inhibitors of this regulation with a high- throughput biological screen. In pursuing these aims, the QS signaling networks of this understudied pathogen will begin to be elucidated and a global assessment of the regulon controlled by this orphan receptor, including potential virulence factors, will be determined. The proposed research is a crucial step towards the long-term objective of understanding QS-regulated virulence in B. mallei and assessing QS as a novel anti-therapeutic target and it will provide important information about the role of orphan QS receptors in bacteria. B. mallei is a category B biothreat agent with few characterized virulence factors and limited treatment options. These studies aim to find BmaRS-controlled virulence factors and identify BmaR5 inhibitors that can be evaluated as novel treatment options. B. mallei animal models are robust and provide an excellent system to assess the role of QS during pathogenesis and for the first time critically evaluate the effectiveness of anti-QS therapeutics in blocking or resolving infections. Characterization of the B. mallei orphan receptor BmaR5 will also contribute to the currently limited understanding of the role of orphan receptors in QS. Also B. mallei are a very close relative of an emerging natural pathogen, B. pseudomallei, and the results of these studies may be directly applicable to QS in B. pseudomallei.